Protein phosphorylation and dephosphorylation are used by cells as a general signal transduction mechanism usually in response to external stimuli. The proteins that carry out these phosphate modifications are enzymes called kinases (phosphorylation) and phosphatases (dephosphorylation). Kinases and phosphatases can be classified into two families depending on the amino acid residue that is (de)phosphorylated: tyrosine kinases (phosphatases) and serine/threonine kinases (phosphatases). Other enzymes that are important in cellular signal transduction pathways include cyclases that can produce cyclic nucleotides such as cyclic AMP (cAMP) and cyclic GMP (cGMP) which are important second messengers, and phosphodiesterases that hydrolyse the cyclic nucleotides to form the corresponding noncyclized nucleotide monophosphates (i.e. AMP and GMP). Due to their important roles in regulating cell function, all these enzymes are important target molecules for the discovery and development of novel pharmaceutical therapeutics.
Traditional methods of measuring the state of phosphorylation of cellular proteins are based on incorporation of radioactive 32P-orthophosphate. The 32P-phosphorylated proteins are separated on a gel and subsequently visualized using a phospho-imager. Alternatively, phosphorylated tyrosine residues may be bound via binding of radiolabelled anti-phosphotyrosine antibodies and detected by immunoassays, for example immunoprecipitation or blotting. Since these methods need to detect radioisotopes, they are time-consuming and also, owing to the safety aspects involved in the handling of radioactive substances, not suitable for ultra high throughput screening (uHTS).
In more recent methods, the radioactive immunoassays are replaced with ELISAs (enzyme-linked immunosorbent assays). These methods use purified substrate proteins or synthetic peptide substrates which have been immobilized to a substrate surface. After a kinase action, the extent of phosphorylation is quantified by the binding of anti-phosphotyrosine antibodies coupled to an enhancer enzyme such as peroxidases, for example, to the phosphorylated immobilized substrates.
Epps. et al. (U.S. Pat. No. 6,203,994) describe a fluorescence-based HTS assay for protein kinases and phosphatases, which makes use of fluorescently labelled phosphorylated reporter molecules and antibodies which bind specifically to the phosphorylated reporter molecules. Binding is measured by means of fluorescence polarization, fluorescence quenching or fluorescence correlation spectroscopy (FCS). This method has the intrinsic disadvantage of only good generic antibodies (e.g. clone PT66, PY20, Sigma) being available for phosphotyrosine substrates. Only a few examples of suitable anti-phosphoserine or anti-threonine antibodies have been reported (e.g. Bader B. et al., Journal of Biomolecular Screening, 6, 255 (2001), Panvera kit no. P2886). These antibodies, however, have the property of recognizing not only phosphoserine but also the neighbouring amino acids as epitope. It is known, however, that kinases function very substrate-specifically and that substrate sequences can differ greatly. Therefore, anti-phosphoserine antibodies cannot be used as generic reagents.
Perkin Elmer (Wallac) provide an assay for tyrosine kinases, which is based on time-resolved fluorescence and an energy transfer from europium chelates to allophycocyanine (see also EP 929 810). Here too, the process is limited essentially to tyrosine kinases, due to the use of antibodies.
Recently, Molecular Devices (U.S. Pat. No. 7,070,921) have provided nanoparticles with charged gallium cations on the surface as a generic binding reagent suitable for phosphorylation reactions to measure kinase and phosphatase activities as well as for nucleotide cyclization and decyclization reactions to measure cyclase and phosphodiesterase activities.